Heat pasturized liquids containing glucosamine

ABSTRACT

The disclosure provides methods of making heat-pasteurized liquids, such as beverages, that contain glucosamine, wherein glucosamine is present in the beverage during the pasteurization process. The disclosure also provides liquids, such as beverages, made by these methods, as well as methods of using the glucosamine supplemented liquids, for example to treat osteoarthritis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/423,119, filed Nov. 1, 2002, and is a continuation-in-part of U.S.patent application Ser. No. 10/685,125 filed Oct. 13, 2003, which is acontinuation-in-part of copending U.S. patent application Ser. No.10/326,549, filed Dec. 19, 2002, which is a continuation of U.S. patentapplication Ser. No. 09/785,695, filed Feb. 16, 2001, and which claimspriority from PCT Application No. PCT/US02/04468, filed Feb. 15, 2002,each of which is incorporated herein by reference.

FIELD

This application relates to heat pasteurized liquids, such as beverages,condiments, and soups that include glucosamine and methods of making andusing such compositions.

BACKGROUND

Beverage supplements can supply consumers with dietary supplements orthe necessary vitamins and minerals specified in the recommended dailyallowances (RDA) provided by the U.S. government. Examples ofnutritionally-balanced beverages are disclosed in U.S. Pat. Nos.3,894,148; 4,309,417; 4,312,856; 4,322,407; 6,432,929; and 6,391,864 aswell as EP Application No. EP 0 681 434.

Dietary supplements for cartilage health and maintenance are effectivein reducing the symptoms of osteoarthritis and,joint pain. Examples ofcartilage supplements include glucosamine (GLCN) hydrochloride, GLCNsulfate, N-acetyl-D-glucosamine (NAG), chondroitin sulfate, hyaluronicacid (which is comprised of a repeating disaccharide ofN-acetyl-D-glucosamine and D-glucuronic acid), and cetyl myristoleate(CM). The most commonly used cartilage supplements are GLCNhydrochloride and GLCN sulfate.

It has been disclosed and the industry has followed the belief thatexposure of GLCN to relatively high temperatures used in food processingapplications, such as pasteurization, would inactivate GLCN (forexample, see U.S. Pat. No. 6,423,929). In attempt to overcome thislimitation, U.S. Pat. No. 6,423,929 teaches that beverages that includeGLCN are prepared using a process that requires two separate heatingsteps to minimize chemical alteration of GLCN. A juice drink base(without GLCN) is prepared using pasteurization at about 195° F. for 42seconds. A separate GLCN water-based solution is prepared at atemperature of below 160° F., such that the GLCN is not inactivated. Thejuice drink base and the GLCN solution are then mixed to form aGLCN-supplemented beverage. Processing a beverage using two differentsolutions at two different temperatures could be relatively expensiveand difficult to implement.

SUMMARY

Disclosed are GLCN-containing liquids, such as beverages, soups, andcondiments, which are treated under high-temperature conditions, such asthose used in heat-pasteurization, without a significant amount of theGLCN being inactivated due to exposure to the high temperature. It hassurprisingly been found that GLCN-supplemented liquids, such asbeverages, can be made under high-temperature heating conditions thatmimic pasteurization without substantially inactivating or degrading theGLCN. In addition, in certain embodiments, the GLCN-supplementedliquids, such as beverages, exposed to high temperatures do not have anunpleasant taste, off color, or off odor.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein and inthe claims, the singular forms “a” or “an” or “the” include pluralreferences unless the context clearly dictates otherwise. For example,reference to “a supplement” includes a plurality of such supplements andreference to “the beverage” includes reference to one or more beveragesand equivalents thereof known to those skilled in the art, and so forth.Similarly, the word “or” is intended to include “and” unless the contextclearly indicates otherwise. Hence “comprising A or B” means includingA, or B, or A and B.

Unless otherwise indicated, all numbers expressing quantities ofingredients, temperatures, time periods, and so forth used in thespecification and claims are to be understood as being modified by theterm “about” whether explicitly stated or not. Accordingly, unlessindicated clearly to the contrary, the numerical parameters set forthare approximations.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs.

Administer: To cause a subject to receive something. As used herein,administration of the disclosed beverages supplemented with GLCN isoral, for example by ingestion.

Beverage: Any drink suitable for ingestion. Includes ready-to-drinkbeverages in their liquid form, such as juice or soda, concentrates, aswell as those in a dry or powered form, such as a tea, instant coffee,or hot chocolate mix. Non-limiting examples of beverages that can besupplemented with GLCN include naturally or artificially flavored fruitor vegetable juices; milk; commercially available sports drinks (sugaror juice based) such as Gatorade®, Powerade®, and Allsport®; soda;Tang®; flavored waters; soy milk; and commercially availablenutritionally-balanced beverages such as Ensure® beverage. A beveragecan be carbonated or non-carbonated. Alcoholic beverages are alsoencompassed by this disclosure, such as wine, wine coolers, maltbeverages and coolers, and beer.

Cartilage dysfunction: A disorder in a subject that results in jointpain or decreased joint mobility, for example arthritis, such asosteoarthritis.

Cartilage supplement: An agent that reduces joint pain, increases jointmobility, reduces swelling, or stimulates joint healing in a subject. Inparticular examples, it is an agent that delays or halts the onset ofosteoartfritis. Examples include, but are not limited to: glucosamine,chondroitin sulfate, hyaluronic acid, chitin, cetyl myristoleate,essential fatty acids, MSM, SAMe, oligoglucosamine, and oligomers ofN-acetyl-D-glucosamine (NAG).

Comprises: A term that means “including.”

Glucosamine (GLCN): As used herein, the term GLCN refers to the variousforms of GLCN, such as agents having the general formula representedbelow, as well as salt complexes and substituted GLCN.

Salts of GLCN include, but are not limited to: citrate, acetate,phosphate, sulfate, chloride, lactate, and gluconate. Examples of GLCNderivatives include glucosamine itself, glucosamine hydrochloride,glucosamine hydroiodide, glucosamine chlorhydrate, glucosamine sulphateand N-acetyl glucosamine.

GLCN can be obtained from any suitable source. In certain examples, GLCNis a GLCN composition that is derived from shellfish, animal cartilage,bacteria, and/or fungal biomass.

In particular examples, GLCN is a GLCN composition that is derived fromfungal biomass containing chitin. Suitable starting materials includemicrobial fungal sources, such as fungal sources derived fromAspergillus sp., Penicillium sp., Mucor sp., and combinations thereof.When GLCN is derived from fungal biomass, it will not pose a hazard topersons who have shellfish allergies because tropomyosin and other suchmuscle-derived proteins are not present in fungal biomass. Therefore,beverages containing GLCN-derived from fungal biomass will be toleratedby persons who have shellfish allergies. In addition, because GLCNderived from fungal biomass is not derived from shellfish (or any animalsource), such GLCN-containing beverages are qualified for kosher statusand may be consumed by strict vegetarians.

GLCN beverage: A beverage that contains GLCN, for example at least about1 mg per serving, at least about 100 mg per serving, at least about 250mg per serving, at least about 500 mg per serving, at least about 750 mgper serving, at least about 1 g per serving, or even at least about 20 gper serving. A pasteurized GLCN beverage is one that includes GLCN inthe beverage when the beverage is exposed to high temperatures, such asthose used in heat pasteurization.

High Temperature: As used herein, refers to temperatures typically usedwhen liquids, such as beverages, are heat pasteurized, for example todestroy undesirable microorganisms.

Particular, non-limiting examples of high temperatures include, but arenot limited to temperatures used in heat pasteurization, for examplehigh temperatures are those at least about 160° F. (about 71° C.), suchas temperatures of at least about 161° F. (about 71.5° C.), at leastabout 180° F. (82° C.), at least 194° F. (about 90° C.), at least about200° F. (about 94° C.), at least about 212° F. (about 100° C.), at leastabout 220° F. (about 104° C.), at least about 280° F. (about 138° C.),or at least about 300° F. (about 149° C.). In particular examples, hightemperatures include temperatures in the range of about 161° F. to about300° F., such at about 161° F. to about 220° F., about 161° F. to about212° F., about 161° F. to about 200° F., 165° F. to about 220° F., 165°F. to about 212° F., about 176° F. to about 220° F., or about 176° F. toabout 212° F.

A GLCN-supplemented liquid, such as a GLCN beverage, can be exposed to ahigh temperature for an amount of time as needed to achieve a desiredeffect, for example to destroy objectionable microorganisms, such as toheat-pasteurize or to bring a GLCN liquid to a boil. Exemplary timesinclude, but are not limited to, at least 15 seconds at a temperature ofat least 160° F., at least 30 seconds at a temperature of at least 160°F., such as at least 20 minutes at about 160° F., at least 20 seconds ata temperature of about 194° F., at least 40 seconds at a temperature ofabout 194° F., or at least 60 seconds at 212° F.

Liquid: A substance in the fluid state of matter having little or nofixed shape but a fixed volume. Exemplary liquids include, but are notlimited to, beverages, soups, yogurt, condiments such as ketchup andmustard, dressing, eggs, syrups, oils, gravies, pudding, and ice creamat a temperature above about 4° C.

Pasteurization: A method used to significantly reduce the presence ofobjectionable organisms (such as bacteria) in liquids (such asbeverages) by exposing the liquid to heat, filtration, or irradiationfor a period of time.

When a GLCN-supplemented liquid is exposed to a high temperature, theterm “heat pasteurization” is used. In some embodiments, heatpasteurized liquids are subsequently cooled quickly to about 38° F. to40° F. to retard the growth of surviving organisms. In particularembodiments, heat pasteurization does not substantially chemically altera liquid, such as beverage or a supplement therein, and does notsubstantially affect the taste or mouthfeel of the liquid. As usedherein, “heat pasteurization” or “heat pasteurized” do not includepasteurization by filtration, or irradiation.

In particular examples, heat pasteurization reduces the number of colonyforming units (cfus) present in a liquid, such as a GLCN beverage, by atleast 50%, such as at least 70%, at least 75%, at least 80%, at least90%, at least 95%, or even at least 98%.

Particular examples of heat pasteurization temperatures are providedabove in under “high temperature”. In some embodiments, heatpasteurization temperatures and incubation times include, but are notlimited to: about 161° F. for about 15 seconds, about 195° F. for about42 seconds (such as about 195±4° F. for about 42±4 seconds), about 200°F. for less than about 40 seconds (such as about 200±5° F. for about40±5 seconds), about 165° F. for about 3 minutes (such as about 165±5°F. for about 180±10 seconds), and at least 280° F. for about 1-2 seconds(for example to ultrapasteurize milk).

If ultrapasteurization is desired, heat pasteurization temperatures canbe increased to about 280° F. or greater (such as about 300° F.), withincubation for a shorter period of time, such as 1-2 seconds.

Preventing disease: A therapeutic intervention that inhibits the fulldevelopment of a disease, for example preventing development ofosteoarthritis in a subject having cartilage dysfunction.

Serving: A serving is the amount of food or beverage a person or animalwould customarily eat in one time. The serving size can often times befound on the Nutrition Facts label on the beverage. Serving sizes arealso shown on the USDA Food Pyramid. For beverages, such as juice orsoda, a serving is usually represented in common household terms, suchas cup or fluid ounce.

Shellfish: A term for mollusks and crustaceans used as food. Exemplaryshellfish include clams, snails, mussels, oysters, scallops, shrimp,lobster, and crayfish. Components of the shell or exoskeleton of theseorganisms can be converted into GLCN using known techniques.

Shellfish protein: A protein present in a shellfish, such as those thatare allergenic in humans having shellfish allergies. Exemplary shellfishproteins include, but are not limited to, shellfish muscle proteins,such as tropomyosin.

Skin disorder: A disease or disorder in a subject that negativelyaffects the skin, and benefits from collagen formation. Examplesinclude, but are not limited to: a wound, wrinkles, and acne. When GLCNis used to treat a skin disorder, GLCN can be introduced into productsused on the skin, such as topical lotions and creams. Alternatively orin addition, GLCN can be introduced into beverages and consumed by asubject in need of treatment or prevention of a skin disorder.

Subject: Living multicellular vertebrate organisms, a category whichincludes both human and veterinary subjects, for example, mammals,rodents, and birds.

Therapeutically Effective Amount: An amount sufficient to achieve adesired biological effect. In one example, it is an amount that iseffective to alleviate or reduce symptoms associated with cartilagedysfunction, such as pain, swelling, and decreased mobility, by morethan a desired amount. In another example, it is an amount that iseffective to stabilize symptoms associated with cartilage dysfunction,such that the symptoms do not worsen. In particular examples, it is aconcentration of GLCN (alone or in combination with other agents) thatis effective to alleviate, reduce, or stabilize symptoms associated withcartilage dysfunction, such as in a subject to whom a GLCN-supplementedliquid, such as a beverage, is administered.

In one example, it is an amount that is effective to alleviate or reducesymptoms associated with a skin disorder, such as promoting the healingof a wound or reducing the appearance of wrinkles, by more than adesired amount. In another example, it is an amount that is effective tostabilize symptoms associated with a skin disorder, such that thesymptoms do not worsen. In particular examples, it is a concentration ofGLCN (alone or in combination with other agents) that is effective toalleviate, reduce, or stabilize symptoms associated with a skindisorder, such as in a subject to whom GLCN is administered.

In one example, a therapeutically effective amount also includes aquantity of GLCN sufficient to achieve a desired effect in a subjectbeing treated. For instance, it can be an amount necessary to improvesigns or symptoms a disease, such as osteoarthritis, a skin disorder, ora wound.

The GLCN-containing beverages disclosed herein have equal application inmedical and veterinary settings. Therefore, the general term “subjectbeing treated” is understood to include all animals (such as humans,apes, dogs, cats, horses, and cows) that require treatment of acartilage dysfunction or skin disorder, such as a wound.

Thermal tolerance: Refers to the ability of GLCN to be exposed to a hightemperature, such as those used in heat pasteurization, without aresulting significant adverse effect on the taste, color, odor, ortexture of a GLCN liquid, such as a beverage, when GLCN is present inthe liquid during pasteurization.

In particular examples, the amount of GLCN present in a GLCN beveragefollowing exposure to a high temperature, demonstrating that GLCN isthermally tolerant, is at least 80%-100%, for example at least 90%-100%, for example at least 93%-100%, for example at least 95%-100%, forexample at least 98%-100%, for example at least 90%-98%, or for exampleat least 93-98%. In other examples, the amount of GLCN present in a GLCNbeverage following pasteurization is at least 80% of the amount of GLCNpresent prior to pasteurization, for example at least 85%, at least 90%,at least 92%, at least 93%, at least 94%, at least 95%, at least 98%, atleast 99%, or even 100% (no loss of GLCN).

Treat: To alleviate or reduce one or more of the symptoms of a disorder,such as a cartilage dysfunction, wound or skin disorder, or to stabilizesuch a condition.

Glucosamine-Supplemented Liquids

Disclosed are GLCN-supplemented liquids, such as beverages, which areexposed to high temperature conditions, such as those using during heatpasteurization of the liquid. The liquid supplemented with GLCN (such asa GLCN beverage) is exposed to one or more high temperatures, and theresulting taste, color, odor, and/or texture of the GLCN liquid is notsignificantly adversely affected. The results disclosed hereindemonstrate that GLCN is heat tolerant and can be placed in liquids,such as beverages, prior to heating to high temperatures, such as thoseused during heat pasteurization. This is surprising given the currentunderstanding of one of ordinary skill in the art that GLCN isinactivated at high temperatures.

The amount of GLCN added to a liquid, such as a beverage, can depend onthe desired concentration of GLCN to be acheived. In certain examples,GLCN is present in the disclosed liquids in amounts effective forpromoting the development of connective tissue in the body, alone or incombination with other agents, such as cartilage promoting agents. Insome embodiments, daily GLCN dosages include at least about 250 mg, atleast about 500 mg, at least about 1000 mg, at least about 2000 mg, oreven about at least 3000 mg. Particular GLCN dosage ranges include, butare not limited to, a range of about 500 mg to about 3000 mg, such asabout 1000 mg to about 2000 mg, such as about 1500 mg of GLCN.

Certain embodiments of the disclosed amounts of GLCN that can beincluded in a liquid, such as a beverage, include, about 0.001 g toabout 20 g GLCN/serving, such as about 0.1 g to about 10 g GLCN/serving,or about 0.5 g to about 0.75 g GLCN/serving. Other examples include atleast 0.01 g GLCN/serving, such as at least about 0.05 g GLCN/serving,at least about 0.1 g GLCN/serving, at least about 0.25 g GLCN/serving,at least about 0.5 g GLCN/serving, at least about 0.75 g GLCN/serving,at least about 1.0 g GLCN/serving, at least about 1.5 g GLCN/serving, atleast about 3.0 g/serving, at least about 5.0 g GLCN/serving, at leastabout 10.0 g GLCN/serving, or even at least about 20.0 g GLCN/serving.In other examples, the amount of GLCN added is about 1 g GLCN/1000 g ofproduct to about 1 g GLCN/0.1 g of product, such as about 1 g GLCN/10 gproduct to about 1 g GLCN/0.5 g product.

In certain embodiments, the GLCN liquids disclosed herein, such asbeverages, are supplemented with one or more other cartilagesupplements, vitamins, minerals, fats, proteins, carbohydrates,sweeteners, organic acids, glucose, unreacted chitin, and glucanconversion materials, such as melanoidins and levulinic acid, orcombinations thereof In addition, other agents that treat cartilagedysfunction or skin disorders can also be included in the disclosedGLCN-supplemented liquids.

Melanoidins are relatively complex, high molecular weight, irregularpolymers and are present in particular embodiments of the GLCN liquids.For example, particular embodiments of the disclosed GLCN liquidsinclude from 0.001 to 15 wt. % melanoidins, or from 0.001 to 1.0 wt. %melanoidins or from 0.01 to 0.1 wt. % melanoidins. Without being tied toany particular theory, melanoidins are likely formed by the conversionof glucans to dextrose to hydroxymethylurfural AHMF) to produce themelanoidins. (The reaction may produce other glucan-derived products andamines from proteins in a biomass source as well as lipids in such asource.) Such a chemical process is the Maillard Reaction.

Levulinic acid (also known as acetyl-propionic acid) is present inparticular embodiments of the disclosed GLCN liquids. Without being tiedto any particular theory, levulinic acid is likely formed when glucansin the fungal biomass are converted to dextrose, which is converted toHMP to finally form formic and levulinic acids. Levulinic acid is anon-hazardous component that is a valuable acidulant used in suchproducts as carbonated and fruit juice beverages, jams, and jellies.Thus, addition of embodiments of the GLCN compositions to such productsprovides an acidulant benefit as well as the benefits provided by theGLCN in the composition. Particular embodiments of the GLCN liquidsinclude from 0.0001 to 1 wt. % levulinic acid, or from 0.001 to 0.7 wt.% levulinic acid or from 0.01 to 0.4 wt. % levulinic acid.

Because the melanoidins and levulinic acid are formed when producing theGLCN compositions according to the disclosed methods, no additionalsteps must be taken to include such components in the compositions.Melanoidins and levulinic acid were not expected in GLCN compositionsderived from shellfish, and analysis of six lots of GLCN derived fromshellfish (obtained from five different suppliers) did not contain anydetectable amounts of melanoidins or levulinic acid.

With reference to Table 1, embodiments of the GLCN compositions usefulfor malting embodiments of the presently disclosed GLCN liquids, such asbeverages, include GLCN derived from fungal biomass and can also includeone or more of the listed components in Table 1, those shown in Table 2and other components as discussed herein. Concentrations of eachcomponent can be within the ranges shown or varied by altering any of avariety of production parameters. TABLE 1 Components that can be presentin a GLCN liquid Representative Representative RepresentativeGlucosamine Embodiment Embodiment Embodiment Composition Percent PercentPercent Components by Weight by Weight by Weight Glucosamine   85-99.8  95-99.8   98-99.8 Melanoidins 0.001-15   0.001-1.0  0.01-0.1 Levulinic Acid 0.0001-1    0.001-0.7  0.01-0.4  Dextrose 0.001-10  0.001-5    0.001-2    Citric Acid 0.001-10   0.01-1.0  0.025-0.5 

Two specific embodiments of the GLCN compositions are set forth in Table2. TABLE 2 Specific embodiments of GLCN liquids Composition *Embodiment1 *Embodiment 2 Component (GP-11) (GP-17C) Ash Content 0.03% 0.02% Si140 ppm 150 ppm Na 10-100 ppm 10-100 ppm K 10-100 ppm 10-100 ppm Ca10-100 ppm 10-100 ppm HCL 0.16% 0.19% Citric Acid 0.045%  0.074% Levulinic Acid 0.39%  0.3% Melanoidins 0.04-0.07% 0.02-0.03%Water-insoluble matter soluble in 0.05% 0.02% gastric juice at ˜40°*Percentages listed are percents by weight

Also disclosed are methods for producing GLCN compositions useful informing embodiments of the presently disclosed GLCN liquids from fungalbiomass sources, including producing such compositions by acidhydrolysis of fungal biomass. Acid hydrolysis breaks ether linkages inthe biomass and deacetylates chitin molecules to generate free GLCN.Acid hydrolysis can break the chitin into GLCN, but leaves the GLCNmolecule substantially intact. Depending upon the acid hydrolysisparameters, acid hydrolysis conditions break down other components (suchas glucans, proteins, and lipids) that exist in the fungal biomass.

In one specific embodiment of the disclosed method for producing GLCNcompositions from fungal biomass, acid hydrolysis is performed bytreating fungal biomass for a relatively long period of time, forexample greater than 4 hours, in a relatively aggressive acid solution.

Chitin-containing fungal biomass may first be reacted in a relativelyaggressive acidic solution. Relatively strong (aggressive) acids can beused to hydrolyze the fungal biomass, including acids of concentrationsless than 50 percent. Acids of concentrations of from about 5 to about25 percent are also suitable. Suitable strong acids includehydrochloric, sulfuric, phosphoric, and citric acid at appropriateconcentrations.

The aggressive acid treatment mixture containing the biomass, acid, andwater is heated and maintained at a relatively elevated temperature. Themixture is usually heated to a temperature at or near its boiling point(typically 90° C. to 106° C.) and maintained under reflux conditions for5 hours or greater, more typically greater than 8 hours, and usuallyless than 16 hours. The reaction may continue long enough to have acomplete breakdown of the chitin, but not so long as to be inefficientor to excessively decompose the GLCN compositions.

Although reaction in the relatively aggressive acid solution produces aGLCN composition, subsequent purification steps can be taken. A firstpurification step may include a separation step, such as filtration, toremove particulate impurities, resulting in a substantially clearsolution of the GLCN composition. The solution contains an embodiment ofGLCN composition as well as small quantities of glucose and othercomponents of the composition. The GLCN composition can be concentratedand some of the acid recovered can be recycled and reused.

In yet other examples, GLCN is a GLCN composition that is derived fromanimal cartilage (for example see U.S. Pat. No. 5,922,692). Suitablestarting materials include vertebrate connective tissue, such as from acow, pig, or chicken. Briefly, to prepare GLCN from cartilage, rawvertebrate connective tissue is disintegrated into an aggregation ofparticles having a substantially homogenous particle size, such as byemulsification, thereby forming liquefied connective tissue. Theliquefied connective tissue is then thermally processed to generate aproduct rich in GLCN.

In particular examples, GLCN is a GLCN composition derived from bacteria(for example see U.S. Pat. No. 6,372,457). For example, GLCN can beproduced by fermentation of a microorganism. Briefly, a microorganismhaving a genetic modification in an amino sugar metabolic pathway iscultured in a fermentation medium. GLCN can then be recovered from thefermentation medium. Exemplary amino sugar metabolic pathways include apathway for transport of glucosamine out of the microorganism or apathway for transport of glucosamine into the microorganism.

In one embodiment, liquids (such as beverages) suitable for use in thepresent disclosure include those pasteurized after GLCN is included inthe liquid. For example, GLCN-supplemented liquids can be prepared usingheat pasteurization at a high temperature, wherein GLCN is present inthe liquid during the heat pasteurization. In certain examples,pasteurization is used to kill a substantial amount of undesirablebacteria.

GLCN-supplemented liquids, such as beverages, can be heat pasteurized ata temperature that will destroy a substantial amount of undesirableorganisms, such as bacteria. Particular non-limiting heat pasteurizationtemperatures include, at least about 160° F., at least about 180° F., atleast about 200° F., at least about 220° F., at least about 280° F., andeven such as at least about 300° F. Particular examples of apasteurization temperature are about 160° F., about 194° F., about 220°F., or even about 280° F. In one example, liquids supplemented with GLCNare exposed to both high temperature and pressure. One example ofpasteurization conditions is about 121° C. at 1 atm for 15 minutes.

In a particular example, GLCN-supplemented liquids are heated to atleast about 170° F. For example, GLCN can be added to coffee, tea, orcocoa. In one example, GLCN is included in a coffee, tea, or cocoamixture (such as a pre-prepared packet) to which boiling or heated water(or other liquid such as milk) is added.

Liquids such as beverages supplemented with GLCN, such as thoseincluding at least about 0.01 g GLCN per serving, and at least about0.001 wt. % levulinic acid are encompassed by this disclosure. Inparticular embodiments, such GLCN-supplemented liquids are at atemperature of at least 160° F. In specific examples, a GLCN liquidcontains no detectable shellfish proteins, such as allergenic shellfishproteins (those causing an allergic reaction in some humans).

Methods of Preparing Glucosamine-Supplemented Liquids

Methods of preparing liquids that include GLCN are disclosed. In oneexample, the method includes heat pasteurizing the liquid, such as abeverage, to a high temperature wherein GLCN is present in the liquidduring the exposure to a high temperature. In another example, themethod includes combining GLCN and a liquid, such as a beverage, therebyforming a GLCN-liquid, then heat pasteurizing the GLCN-liquid at a hightemperature.

In certain embodiments, the amount of GLCN present in the GLCN-liquidprior to the heat pasteurization is substantially similar to an amountof GLCN in the GLCN-liquid after heat pasteurizing. In one example, theamount of GLCN in the GLCN liquid after heat-pasteurizing is at leastabout 80% of the amount of GLCN in the GLCN liquid prior toheat-pasteurizing, such as at least about 90%, at least about 95%, atleast about 98%, or even about at least 100%.

Treatment Using Glucosamine

A method of treating a cartilage dysfunction in a subject byadministering the disclosed GLCN liquids is disclosed. In some examples,treatment alleviates or reduces the symptoms of cartilage dysfunction,such as increases joint mobility, reduces pain, or reduces swelling inthe subject. In certain embodiments, treatment stabilizes the symptomsof cartilage dysfunction, such that the cartilage dysfunction is notexacerbated. Examples of cartilage dysfunction include, but are notlimited to, joint pain and osteoarthritis.

Also disclosed are methods of treating a skin disorder in a subject byadministering the disclosed GLCN liquids, such as beverages, to asubject. In some examples, treatment alleviates or reduces the symptomsof a skin disorder, such as promotes wound healing in the subject. Insome examples, treatment stabilizes the symptoms of a skin disorder,such that the skin disorder is not exacerbated. Examples of skindisorders include, but are not limited to, wounds and wrinkles.

A method for treating food allergies in a subject by administering thedisclosed GLCN liquids to the subject is disclosed. In some examples,treatment alleviates or reduces the symptoms of a food allergy, such asreduces the inflammatory response to the food in the subject. In someexamples, treatment stabilizes the symptoms of a food allergy, such thatthe food allergy is not exacerbated.

The subject treated can be a human or veterinary subject suffering fromcartilage dysfunction, skin disorder or food allergy (for example see WO93/14766A1). An effective amount of GLCN can be administered in a singleserving, or in several servings, for example daily, during a course oftreatment. However, the effective amount can be dependent on the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration.

A typical amount of GLCN delivered in dietary supplement products isabout 1500 mg per day, in a single or in multiple administrations. Forexample, if the subject was to receive multiple administrations in asingle day, the subject might receive three servings of GLCN, eachcontaining about 500 mg GLCN. GLCN can be administered at about at leastabout 0.001 g per day. In one example, GLCN is administered at about 750mg GLCN per day. In other examples, GLCN is administered at about atleast 10 mg per day, such as about at least 50 mg per day, about atleast 100 mg per day, about at least 250 mg per day, about at least 500mg per day, about at least 750 mg per day, about at least 1.0 g per day,about at least 1.5 g per day, about at least 3.0 g per day, about atleast 5.0 g per day, about at least 10.0 g per day, or even about atleast 20.0 g per day.

EXAMPLE 1 Allsport® Sports Drink

Allsport® sports drink (The Monarch Company, Atlanta, Ga.) was used asthe basis for incorporation of samples. Citrus Slam flavor was chosenbecause of its light color, which allows panelists observe color change.According to the manufacturer, a 20-ounce bottle of Allsport® contains2.5 servings.

For servings that included GLCN, the serving had 0.75 g GLCN added,which reflects a typical amount of GLCN delivered in dietary supplementproducts. Therefore, 1.875 g (0.75 g/serving GLCN×2.5servings/bottle=1.875 g GLCN/bottle) of GLCN was added to a 20-ouncebottle of Allsport®. After the bottles were prepared, the contents wereheated to 195±4° F. for 42±4 seconds to simulate a heat pasteurizationstep. Samples were heated in a 1000 watt Amana Radarange microwave ovenin foam cups for the same amount of time. For a room temperaturebeverage, 150 seconds of heating time was necessary, and for arefrigerated beverage, 180 seconds was required. The beverages werecooled in their original bottles, ¾ submerged in an icewater bath, andhand rotated to simulate flash cooling.

Samples were tested within one day of preparing. Samples were testedusing a “Difference From Control” test, using a marked control, and ablind control was included. Panelists were asked to compare each sampleto the control and comment. Panelists received the followinginstructions:

-   -   1. Taste the control sample first.    -   2. Compare all other samples to the control and write        descriptors in the table.

3. Numerically rate whether the samples are better or worse than thecontrol, using this scale: −5 −4 −3 −2 −1 0 +1 +2 +3 +4 +5 Worse Same asBetter than control than control control

-   -   4. Please do not discuss the results with any other panelists        until all sheets are turned in.

The 12 Panelists' results are shown in Table 3. TABLE 3 Results ofAllsport ® sports drink.* Mouthfeel/ Sample Color Odor texture TasteComments (other than “same”) Control +0.1 0 +0.4 −0.1 Less sour thanothers, sweeter (2), more of a carbonated feel, stronger odor (2), moretangerine taste, mouthfeel thicker Fungal 0 −0.3 +0.1 −0.3After/off-taste (2), “vitamin” like taste, GLCN sweeter, less odor,sour, yucky Shellfish −0.1 −0.3 −0.2 −0.7 Sweeter (2), sour, crystals ontop, less odor, GLCN mouthfeel thicker, less tangy, flat and yucky*Results are the average rounded to nearest tenth.

As shown in Table 3, it was difficult for the panelists to ascertaindifferences between the samples. The control was not clearlydifferentiated from GLCN. These findings demonstrate that taste andother factors are not adversely affected when GLCN is used in a hightemperature beverage application, such as those that require heatpasteurization. That is, the presence of GLCN did not significantlyaffect the taste of the final GLCN beverage, even though GLCN wasexposed to a high temperature.

EXAMPLE 2 Libby's White Grape Juicy Juice®

Libby's White Grape Juicy Juice® was used as the basis for incorporationof samples. White Grape was chosen due to its light color, to helppanelists observe any color change. According to the manufacturer, a 64ounce bottle contains 8 servings of 8 ounces each.

For servings that included GLCN, the serving (8 ounces) had 0.75 g GLCNadded, which reflects a typical amount of GLCN delivered in dietarysupplement products. 16-ounce portions of juice were used. To theappropriate batches, 1.5 g of GLCN (16 ounces is two servings, and 2servings×0.75 g/serving=1.5 g) was added. After preparing the samples,they were heated to 195∓4° F. in a microwave, held at that temperaturefor 42 seconds to simulate heat pasteurization, then cooled as describedin Example 1.

Samples were tested according to the methodology and instructions inExample 1. The 12 Panelists' results are shown in Table 4. TABLE 4Libby's White Grape Juicy Juice ® results.* Mouthfeel/ Sample Color Odortexture Taste Comments (other than “same”) Control +0.1 0 0 0 Slight offtaste, more lingering taste Fungal −0.2 −0.3 −0.3 −0.6 Darker (2),thicker mouthfeel (2), sweeter (2), GLCN stale/weaker (2), smooth,soapy, lasting aftertaste, tart, odor masked Shellfish −0.4 −0.1 −0.3−1.1 Darker (3), thicker mouthfeel, sweeter (2), GLCN soapy (2), tart,bitter, less sweet, less fruity (2)*Results are the average rounded to nearest tenth.

The data shown in Table 4 is validated by identification of the control,based on the control score of near “0” in all categories. As shown inTable 4, it was difficult for the panelists to ascertain differencesbetween the samples. These results further demonstrate that taste andother factors are not adversely affected when GLCN is used in a hightemperature beverage application, such as those including a heatpasteurization step.

EXAMPLE 3 Hy-Vee® Healthy Recipe Tomato Soup

Hy-Vee® Healthy Recipe Tomato Soup was used as the basis forincorporation of samples. Tomato soup was chosen for its even texture,and “healthy” aspect when compared to other soups. According to themanufacturer, one can makes 2.5 servings.

For servings that included GLCN, the serving had 0.75 g GLCN added,which reflects a typical amount of GLCN delivered in dietary supplementproducts. Each batch prepared 2.5 servings. For batches 2-4, 1.875 gGLCN was added (0.75 g GLCN/serving×2.5 servings=1.875 g GLCN/batch).The recipe on the can was used. The soup can contents were poured into amicrowaveable container, one can of water slowly stirred in, and GLCNadded (where applicable) and the mixture stirred well. The container wascovered with vented plastic wrap (turn back edge of wrap to form smallopening for steam to escape), and the mixture heated in a 1000 wattAmana Radarange at high power for 2 minutes. The sample was not allowedto come to a boil.

Samples were tested according to the methodology and instructions inExample 1. The 8 Panelists' results are shown in Table 5. TABLE 5Hy-Vee ® Healthy Recipe Tomato Soup results.* Mouthfeel/ Sample ColorOdor texture Taste Comments (other than “same”) Control −0.2 0 −0.1 +0.2less tangy (2), lighter in color Fungal 0 0 −0.1 −0.3 Off flavor, alittle metallic, flat/sour taste but GLCN barely perceptible, more tangyw/ acidic aftertaste (like black pepper bad been added), milder, maybesweeter, less tangy aftertaste Shellfish −0.2 0 −0.3 −0.6 Slight offflavor, dirty odor, flat/sour taste but GLCN barely perceptible, lighterin color, off aftertaste, bitter, chalky in texture, sour*Results are the average rounded to nearest tenth.

The results shown in Table 5 are validated by identification of thecontrol; based on the control score of near “0” in all categories. Itwas difficult for the panelists to ascertain differences between thesamples. These results demonstrate that taste and other factors are notadversely affected when GLCN is used in a high temperature foodapplication, such as those that include heating or boiling prior toconsumption.

EXAMPLE 4 Lemonade

Lemonade made from scratch was used as the basis for incorporation ofsamples. To prepare the lemonade, the following recipe was used: 10.8 gdry dextrose, 82.0 g 42 high fructose corn syrup (HFCS), 5.6 g citricacid, 0.5 g sodium citrate, 5.7 g GLCN or NAG, then add water to onequart. Lemonade was prepared for sensory testing as follows:

-   -   Batch 1. Control Lemonade (no fungal glucosamine)    -   Batch 2. Lemonade+fungal glucosamine (pH 3-4)    -   Batch 3. Lemonade+fungal glucosamine (pH 3-4) heated to 71° C.        for 20 minutes    -   Batch 4. Lemonade+fungal glucosamine (pH 3-4) heated to 90° C.        for 20 seconds, flash cooled in ice water bath    -   Batch 5. Lemonade+fungal glucosamine (pH 3-4) boiled on stove        for 5 minutes, flash cooled in ice water bath

The recipe prepared one quart, so each batch consisted of 32 ounces, orfour 8-ounce servings. For servings that included GLCN, the serving (8ounces) had 1.425 g GLCN (1.425 g/serving×4 servings/batch=5.7 g/batch),which reflects a typical amount of GLCN delivered in dietary supplementproducts.

Samples were tested according to the methodology and instructions inExample 1. The four Panelists' results are shown in Table 6. TABLE 6Results of Lemonade.* Actual 30 sec Sensory Panel Sample Temp ° C.Initial pH Temp ° C. Mouthfeel Taste Observations Control Not heated3.13 N/A 0.5 0 Tart, sweet, bitter (2), astringent GLCN not Not heated3.10 N/A 0.8 2.8 More mild, more heated sour than sweet, sweeter (2)GLCN 71 3.03 67 1.3 1.5 Stronger sweet (2), 71° C. tart, less sweet,more mild GLCN 90 3.05 74 1.3 1.8 Chemical taste, sweet 90° C. (2), flattaste (2), strong taste GLCN 97 3.30 73 0.8 −1.0 Less sweet, boiledcardboard essence, off taste, less flavor, bitter*Values shown are the average rounded to nearest tenth.

As shown in Table 6, these results further demonstrate that taste andmouthfeel are not adversely affected when GLCN is used in a hightemperature food application, such as those that include heating or evenboiling prior to consumption. In several samples, the presence of GLCNimproved taste and mouthfeel relative to the control samples.

EXAMPLE 5 Determination of the Amount of GLCN Present Following Heating

To determine the amount of GLCN that remains following exposing GLCNbeverages to a high temperature, such as those used in pasteurization,the following methods were used.

A beverage sample as described in the preceding examples containing S to20 mg of GLCN was dispersed in 25 g of 1.0 N HCl in a 50-mLpolypropylene centrifuge tube and capped tightly. The sample was mixedfor 30 seconds using a vortex mixer, then placed in a water bath at 37°C. The sample was removed from the water bath at 15-minute intervals,mixed for 30 seconds on a vortex mixer, then returned to the water bath.This cycle was repeated until the sample had been in the water bath forone hour.

After heating, the sample was mixed for 30 seconds on a vortex mixer,then centrifuged for 10 minutes to separate the liquid and solid phases.Fats, oils or lipids in the sample formed a third layer at the top ofthe tube. A 1-g aliquot of the aqueous sample portion was diluted100-fold with deionized water, then transferred to an autosampler vialwith filter cap.

The free glucosamine in prepared samples was determined using highperformance anion-exchange chromatography with pulsed amperometricdetection (IPAEC-PAD). The system included an EG40 eluent generator,GP50 gradient pump, AS40 autosampler, LC25 column oven, and ED40electrochemical detector, all produced by Dionex Corporation (Sunnyvale,Calif.).

The method was adapted from Dionex Corporation Technical Note 40. ADionex CarboPac PA-20 column was used in place of the PA-10 described inthe Technical Note. The eluent was 8 mM KOH at 0.5 mL/min. The columnand detector were maintained at 30° C. The injection volume was 10 μL.The standard was glucosamine hydrochloride at 10.8 mg/L. Fermentationbroth samples were diluted five-fold with deionized water, ASTM Type II,and filtered through 0.2 μ vial filters in the autosampler. Multiplestandards were analyzed before and after each sample set. The resultsare shown in Table 7. TABLE 7 Percent of GLCN recovery followingheating. Sample Temp ° C. Time % GlcNHCl Recovered Lemonade Roomtemperature 102 Lemonade 90 20 sec 101 Lemonade 71 20 min 101 Lemonadeboil  5 min 100 Grape juice boil  5 min 102

As shown in Table 7, there was little degradation of GLCN when thebeverages were boiled. The amount of recovery for GLCN was about 100%.Therefore, in contrast to the previous understanding of those skilled inthe art, GLCN is not substantially degraded or inactivated followingexposures to high temperatures, such as those used in heatpasteurization.

In view of the many possible embodiments to which the principles of thisdisclosure may be applied, it should be recognized that the illustratedembodiments are only particular examples of the disclosure and shouldnot be taken as a limitation on the scope of the disclosure. Rather, thescope of the disclosure is in accord with the following claims. Wetherefore claim all that comes within the scope and spirit of theseclaims.

1. A method for making a beverage comprising: providing a beverage;providing an amount of glucosamine (GLCN); mixing the beverage and theGLCN, thereby forming a GLCN beverage; and heat-pasteurizing the GLCNbeverage at a high temperature for a time sufficient to reduce colonyforming units (cfu) by at least about 50%.
 2. The method of claim 1,wherein heat-pasteurizing the GLCN beverage comprises heating the GLCNbeverage to at least about 160° F.
 3. The method of claim 1, whereinheat-pasteurizing the GLCN beverage comprises heating the GLCN beverageto at least about 200° F.
 4. The method of claim 1, whereinheat-pasteurizing the GLCN beverage comprises heating the GLCN beverageto a temperature in a range of from about 160° F. to about 300° F.: 5.The method of claim 1, wherein the GLCN beverage is heat-pasteurized fora time period from about 1 second to about 5 minutes.
 6. The method ofclaim 1, wherein the amount of GLCN added to the beverage is at leastabout 0.1 g GLCN per serving
 7. The method of claim 6, wherein theamount of GLCN added to the beverage is at least about 0.25 g GLCN perserving.
 8. A method for making a beverage comprising: providing abeverage; providing a first amount of GLCN; mixing the beverage and theGLCN, thereby forming a GLCN beverage; and heat-pasteurizing theGLCN-beverage, wherein the amount of GLCN in the GLCN beverage prior toheat-pasteurizing is substantially similar to a second amount of GLCN inthe GLCN beverage after heat-pasteurizing.
 9. The method of claim 8,wherein the second amount of GLCN in the GLCN beverage afterheat-pasteurizing is at least about 80% of the first amount of GLCN inthe GLCN beverage prior to heat-pasteurizing.
 10. The method of claim 1,wherein the GLCN is derived from a fungal biomass containing chitin. 11.A beverage made by the method claim
 1. 12. A beverage comprising: atleast about 0.01 g per serving of GLCN; and at least about 0.0001 wt. %levulinic acid.
 13. A beverage comprising: at least about 0.01 g perserving of GLCN; and at least about 0.0001 wt. % melanoidins.
 14. Thebeverage of claim 13, wherein the beverage does not contain shellfishproteins.
 15. A beverage comprising: at least about 0.01 g per servingof GLCN; at least about 0.0001 wt. % levulinic acid and/or at leastabout 0.0001 wt. % melanoidins; and wherein the beverage is at leastabout 160° F.
 16. The beverage of claim 13, wherein the beveragecomprises at least about 0.25 g GLCN per serving.
 17. The beverage ofclaim 15, wherein the beverage comprises at least about 0.25 g GLCN perserving.